Our principal objective is to develop and apply new, improved methods for analysis of biological materials that require mass spectrometric (MS) analysis, but which have not previously been amenable to such analysis by reason of volatility, thermal lability or charge state. Our basic approach involves a direct interface of high performance liquid chromatography (HPLC or LC) effluent with the mass spectrometer source, and permits use of conventional solvent flow rates and buffered aqueous solvents (1 m./min 0.1 M ammonium acetate). Ions are desorbed directly from vapor droplets that are heated rapidly in passage from the HPLC capillary through the ion source by a mechanism that resembles other desorption techniques (field desorption, laser desorption, fast atom bombardment (FAB). Thermospray LC/MS has the important advantages over these other methods of a) a chromatographic inlet, b) applicability to analysis of mixtures, and c) simplicity of sample preparation. Recent applications include: 1) Identification and quantification of novel fatty acid conjugates of carnitine in subjects with Reye's Syndrome, organic acidurias and valproic acid toxicity; 2) Separation and identification of the biosynthetic pathway of hypersine, an unusual basic amino acid that is a conjugate of spermidine and lysine, showing that the E-nitrogen comes from lysine; and 3) Separation and quantification of choline (Ch) and acetylcholine (ACh) from mouse brain. Sensitivity is comparable to electrochemical detection methods, but this work represents the first direct measurement of ACh. Quantitites of Ch and ACh found are comparable to earlier reports and show the biological variation expected; 4) Separation and quantification of catecholamines prior to working on a biosynthetic pathway problem; and 5) Separation and quantification of cortisol prior to developing a rapid clinical measurement for cortisol MCR.